Organotypic intestinal culture and methods of use thereof

ABSTRACT

An organotypic culture comprises an artificial stroma overlayed with epithelial cells isolated from a human colon or intestine. The stroma comprises a mixture of collagen and human fibroblasts isolated from a human colon or intestine. The culture contains a factor that binds the IGF-1 receptor, a factor that binds the EGF receptor, and a factor that binds the LIF receptor. These factors may be added exogenously to the culture via medium or may be expressed by various recombinantly engineered cell types in the culture. The organotypic culture can result in growth that is in situ-like or emphasizes other physiological or morphological states, depending on the balance of factors in the growth media. The organotypic culture may be used in methods for screening of therapeutic, carcinogenic, or growth enhancement factors, or for treating intestinal injuries by applying to the site of an injury the intact culture or the components thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage of PCT/US02/26663, filed Aug. 22,2002, which claims the benefit of the priority of US Provisional PatentApplication No. 60/314,111, filed Aug. 23, 2001.

Aspects of this invention were supported by the National Institutes ofHealth grant Nos. CA74294, PK50306, and CAI 08185. The United Statesgovernment may have certain rights in this invention.

BACKGROUND OF THE INVENTION

The normal human colonic epithelium undergoes continuous cycle ofrenewal with a dynamic equilibrium between proliferation,differentiation and apoptosis. Within the base of each crypt (i.e., adeep indentation formed by involutions of the colonic epithelium), yetto be identified stem cells give rise to progenitor cells that dividerapidly four to six times before differentiation. In the mouseintestine, up to 60% of the approximately 250 epithelial cells in asingle crypt divide twice daily, yielding up to 260 new cells. Thus, theintestinal epithelium harbors one of the most rapidly dividing celltypes in any mammalian organ. Polarized cells migrate along the crypt'sbasement membrane towards the apical surface of colonic villi, where thecells undergo differentiation as indicated by shifts in cytoskeletalmarkers such as cytokeratins, cytoplasmic carbonic anhydrase isozyme II,and the brush border enzyme alkaline phosphatase.

Induction of differentiation is incompletely understood, but appears toresult from a concerted interplay between growth factors produced byepithelial and stromal cells and signals from basement membranecomponents. The basement membrane is synthesized by both epithelial andmesenchymal cells, and it contains numerous fenestrations through whichprocesses of myofibroblasts and/or epithelial cells extend. Little isknown about the role of fibroblasts in modulating proliferation anddifferentiation of colonic epithelial cells except that fibroblasts canprolong their survival. Heterologous cross-talk between epithelial andmesenchyrnal compartments involves basement membrane molecules andparacrine factors. The mesenchymal cells apparently produce as yetundefined growth factors for the epithelial cells.

Normal human colonic epithelial cells have been difficult to maintain invitro. Thus knowledge about intestinal cell regulation has been derivedfrom studies with cell cultures isolated from experimental animals andhuman colon cancer-derived cell lines. Normal epithelial cells surviveonly a few days in culture, which has limited studies of proliferationand differentiation. For example, in Whitehead et al's culture(Whitehead R. H. et al, 1999 Gastroenterology, 117:858–865), normaladult colonic crypt cells were embedded into an acellular collagen gelmatrix over a feeder layer of bovine aortic endothelial cells, and grownas isolated islands of cells, which increased their survival for up to16 days.

Normal human cells grown as isolated cultures in monolayer lose manycharacteristics of those in situ and often resemble the phenotype ofcancer cells. For example, colonic cells immortalized with viraloncogenes lose their typical epithelial morphology and neither polarizenor differentiate, limiting the use of their usefulness for biologicalstudies.

In contrast, cells in a tissue-like context maintain a similar phenotypeas those growing in situ. Organotypic reconstructs and cultures canserve as replacement organs, as models for the study of the basicbiology of organs, and as screening systems for development of drugs, toidentify drug candidates as well as to observe candidate drug activity,such as its transport into organs, or dosage requirements. Obviously,the most useful organotypic cell reconstructs and cultures have a longshelf life and allow the component cells to maintain their normalcellular activities and morphologies and the ability to function withinthe organ.

Organotypic culture models for esophagus, bladder, pancreatic duct,breast, lung, liver, and human skin have all been used for studies oftissue physiology, drug delivery and transformation. The cells in thoseorganotypic cultures retain many of the functions they had in situ. Forexample, normal human melanocytes in the epidermis of an organotypicskin culture were shown to develop close adhesive and gap junctionalcommunications with basal layer keratinocytes.

In contrast to cells of most other organs, normal human colon cells havebeen difficult to maintain in vitro. Currently available normal humanintestinal epithelial cells are derived from the small intestine andexhibit undifferentiated features, while differentiated enterocytesremain in culture for only 10–12 days. Models of human intestine inculture are not suitable for studies of proliferation anddifferentiation. The cultured cells survive for only a few days.Co-culture of intestinal epithelial cells with fibroblasts ormyofibroblasts could prolong survival. To improve survival,immortalization of colonic cells with genes from oncogenic viruses hasbeen attempted. However, the transformed cells lost their typicalepithelial morphology and did not polarize or differentiate.

There remains a need in the art for compositions and methods thatprovide a useful source of normal human intestinal epithelial cellswhich maintains in situ-like properties for use in studies of colonbiology, screening for drug absorption and efficacy and for therapeuticuses, such as in transplantation or the treatment of colon lesions.

SUMMARY OF THE INVENTION

In one aspect, this invention provides an organotypic culture comprisingan artificial stroma comprising a mixture of collagen and humanfibroblasts isolated from a human colon or intestine, the stromaoverlayed with epithelial cells isolated from a human colon orintestine. Present in the culture are at least one growth factor thatbinds the insulin growth factor-1 (IGF-1) receptor, at least one growthfactor that binds the epidermal growth factor (EGF) receptor, and atleast one growth factor that binds the leukemia inhibitory factor (LIF)receptor. Desirably this organotypic culture resembles the in situ colonor small intestine tissue. In another embodiment, cells of theorganotypic culture are in specific pre-hemostatic stages.

In another aspect, the invention provides a culture medium suitable forgrowth of an organotypic culture of claim 1 comprising a base media, atleast one of insulin or IGF-1, at least one of EGF-1 or tumor growthfactor (TGF)-alpha, and LIF. In another embodiment, the medium includesa base media, 1% fetal calf serum (FCS) and/or transferrin, and at leastone factor selected from among insulin or IGF-1, EGF or TGF-alpha,endothelin-3 (ET-3), hepatocyte growth factor (HGF), LIF, stem cellfactor (SCF), and autocrine mobility factor (AMF). Still additionalembodiments are disclosed below.

In still another aspect, the invention provides a method of preparing anorganotypic culture as above-described. This method involves assemblingan artificial stroma by mixing collagen and fibroblasts; and seeding theartificial stroma with epithelial cells in the presence of at least onegrowth factor that binds the IGF-1 receptor, at least one growth factorthat binds the EGF receptor, and at least one growth factor that bindsthe LIF receptor.

In yet another aspect, the invention provides a method of in vitroscreening of an agent comprising contacting an above-describedorganotypic culture with a selected agent in a vessel, and observing theeffect of the agent upon the culture. Depending upon the desired effectsought, this method enables one to select from among many agents, anagent suitable as e.g., a drug candidate, or an agent that affects cellreplication, proliferation or differentiation in a desirable way. Thisscreening method permits the identification of agents that would affectwound repair, agents that are carcinogens, and/or agents that absorb orcross membrane transport into tissue.

In still a further aspect, the invention provides a method for screeningan agent for repairing effect on intestinal epithelial cell injury. Thismethod involves disrupting the layer of epithelial cells on anabove-described organotypic culture; and contacting the site ofdisruption with the selected agent; The effects of the agent on therepair of the epithelial cell layer are observed, thereby enablingselection of an agent which promotes repair of the epithelial cell layerand/or is capable of repairing the injury.

In yet another aspect, the invention involves a method for enhancingepithelial cell repair at an in vivo site of intestinal or colonicinjury. This method involves delivering to the site of the injury atleast one of collagen, fibroblasts, a growth factor that binds the IGF-1receptor, a growth factor that binds the EGF receptor, and a growthfactor that binds the LIF receptor, or a combination of these factors.In one embodiment, the fibroblasts may be recombinantly engineered toexpress one or more of the other factors.

In still another aspect, the invention involves a method of treating anintestinal wound by placing an organotypic culture as defined herein onan intestinal wound in a patient.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of the embodimentsthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph plotting diameter (mm) of constriction of collagenover time of incubation (mean±SD of 8 samples) in a colonic culturepreparation including fetal colon (18 weeks' gestation) after isolationof mucosa and after the epithelial layer is stripped, including thebottom of crypts.

FIG. 2A is a bar graph illustrating the results of ³H-thymidineincorporation assays on organotypic cultures of the invention grown invariously supplemented media, as discussed in Example 4. Significantstimulation of cells in base medium with LIF (BM+LIF) is compared withbase medium (BM) only. Incorporation was measured after 2 days whencells were cultured on matrix without fibroblasts. Results are mean(±SD) median of 4 experiments.

FIG. 2B is a bar graph illustrating that the number of colonicepithelial cells is significantly higher in LIF-containing base medium.Results are mean (±SD) from 360 fields.

FIG. 2C is a bar graph illustrating that numbers of both BrdU-positiveand total epithelial cells were significantly higher in LIF-stimulatedcultures, whereas there were no differences in fibroblast numbers.

FIG. 3A is a bar graph illustrating differentiated cells in organotypiccolon cultures, showing a percentage of goblet cells among total cellsand per area, indicating lack of stimulation by LIF. There was a trend(p=0.10) toward increased absolute numbers of goblet cells inLIF-stimulated cultures.

FIG. 3B is a bar graph illustrating differentiated cells in organotypiccolon cultures, showing a decrease of total enteroendocrine cell numbersin LIF-containing medium.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a novel organotypic culture model, also referredto as an intestinal reconstruct, that mimics in situ conditions of thenormal human colon. This culture is thus useful in the investigation ofcolon epithelial cell proliferation and differentiation, and permits thedissection of the role of individual growth factors in the cross-talkbetween the epithelial and mesenchyrnal compartments that form thenormal intestinal wall. Thus this organotypic culture is useful forscreening of agents which may have therapeutic or toxic effects on theintestinal wall, as well as other research and therapeutic usesdescribed below.

I. The Organotypic Culture of the Invention

In the normal intestine, both epithelial cells and myofibroblastscontribute to formation of the basement membrane (Kedinger, M. et al,1998. Ann. NY. Acad. Sci. 859: 1–17). The organotypic culture of thisinvention comprises an artificial stroma (also known as a stromalreconstruct or stromal matrix) overlayed or seeded with epithelial cellsisolated from a human colon or human intestine. This culture furthercontains at least one growth factor that binds the insulin growthfactor-1 (IGF-1) receptor, at least one growth factor that binds theepidermal growth factor (EGF) receptor, and at least one growth factorthat binds the leukemia inhibitory factor (LIF) receptor. A reconstructcomprising either type of epithelial cells is referred herein as anintestinal reconstruct. This combination of all three components, i.e.stromal cells, collagen, and colonic or small intestine epithelial cellsgrown in contact with the collagen and stromal cells is important inallowing the growth of the epithelial cells and assembly of an in situlike reconstruct. The reconstruct can be grown in a media whichcomprises base media supplied with specific subsets of factors, whichallows for identification of agents that effect specific morphologicaland interactive phenomena of cells within the reconstruct.

A. The Artificial Stroma

The artificial stroma is a matrix formed by a mixture of collagen andhuman fibroblasts isolated from a human colon or intestine. Optionally,the stromal reconstruct further comprises smooth muscle cells. Stilloptionally, the stromal reconstruct may contain other types of cells,such as neurons, perycites, endothelial cells and macrophages, amongothers. The artificial stroma provides a collagen substrate with stromalcells to closely mimic physiological conditions. Thus, in thethree-dimensional organotypic culture, the artificial stroma permits theisolated human fetal colonic epithelial cells to be maintained in theirnative milieu. The fibroblasts constrict the collagen in the artificialstroma, allowing the colonic epithelial cells to migrate, proliferateand differentiate.

Collagen from any source can be used in the artificial stroma. In oneembodiment collagen type I is used in the stroma; in another embodimentcollagen type III is used in the stroma. Collagen types I and III arethe major types of collagen present in the normal stroma and intestine.In one embodiment, the collagen used in the artificial stroma is humancollagen. In another embodiment, another mammalian collagen may be usedto form the artificial stroma, such as bovine tendon acid-extractedcollagen (Organogenesis, Canton, Mass.). Another commercially availablecollagen which can be used in this invention is rat tail collagen(Collaborative Research Products).

Any human fibroblasts are useful in admixture with the collagen to formthe artificial stroma. In one embodiment, the fibroblasts are humancolonic fibroblasts. In a specific embodiment, the fibroblasts are adulthuman colon fibroblasts. In another embodiment the fibroblasts are fetalhuman colon fibroblasts. In another embodiment the fibroblasts are adulthuman small intestinal fibroblasts. In still another embodiment, thefibroblasts are fetal human small intestinal fibroblasts. Still anothersource of fibroblasts are human fibroblast stem cells, which may bederived from bone marrow. The fibroblasts can be newly isolated from theabove sources or the fibroblast cells can be from an established cellline.

Methods of establishing and propagation of mammalian fibroblast celllines are well known in the art (Bell et al., 1993 J. Invest Dermat., 81Suppl.: 2s–10s). A few such cell lines were produced from fetus explantsas described in the Examples. One such line used as a source forfibroblasts for the artificial stroma of this invention is FFC331. Itwas propagated on Dulbecco's modified minimum essential medium (DMEM,GIBCO BRL, Rockviile, Md.) supplemented with 10% fetal calf serum (FCS,Cansera, Rexdale, Ontario, Canada) and antibiotics. Cultures were usedup to passage 10. Use of cells from cell lines that did not undergoexcessive passages is preferred. Other sources of suitable cell linesmay be obtained from commercial or institutional laboratories andfacilities, such as the American Type Cell Culture, Manassass, Va.

In one embodiment of the artificial stroma, human smooth muscle cellsare also embedded in the collagen, along with the fibroblasts. Humansmooth muscle cells from any organ or tissue can be used, for example,from the abdomen or the vascular system. However, vascular smooth musclecells are preferred. The cells can be freshly isolated from adulthumans, from fetal cell sources, or as stem cells from the bone marrow.Alternatively, the cells can be obtained from an established cell line.One such line predominantly used as a source of smooth muscle cells wasHIAS119 (Dr. E. Levine, The Wistar Institute). The HIAS119 cells wereisolated from human large vessels and maintained in medium M199,supplemented with 10% FCS, 2 mM L-glutamine, and 50 g/ml of bovinehypothalamic extract (Sorger, T. et al., 1995 In Vitro Cell Dev. Biol.Anim., 31:671–683). Other sources of suitable cell lines may be obtainedfrom commercial or institutional laboratories and facilities, such asthe American Type Cell Culture, Manassass, Va.

The ratio of fibroblast and smooth muscle cells (if used) is subject toa large degree of variability. Preferably, the ratio offibroblast:smooth muscle cells is about 1:1 or higher. In a particularlypreferred embodiment, the ratio is 10:1 fibroblasts:smooth muscle cells.

In another embodiment, the fibroblasts and optionally the smooth musclecells for admixture into the collagen matrix to form the artificialstroma are genetically engineered to permit the cells to overexpress oneor more factors that are desirable for growth and maintenance of theorganotypic culture of this invention. The techniques, vectors andfactors useful for the generation of such fibroblasts (and optionallysmooth muscle cells) are described in detail below, under the heading“Manipulation of Cells of the Organotypic Culture”.

For preparation of the artificial stroma, the collagen is suspended insuitable medium. In one embodiment, used in the Examples below, themedium is DMEM supplemented with Vitamin C (Sigma) at 50 M/liter,L-glutamine (GIBCO; BRL) at 1.66 mM and 1% fetal calf serum (FCS) to afinal concentration of 0.9 to 1.1 mg/ml. The suspension is neutralized,preferably to about pH 7.0. In one embodiment, a pH of 7.2 is used.Before the collagen gel hardens (usually within about 15 minutes),fibroblasts and optional smooth muscle cells are added. The stromal(i.e., fibroblast) cells are added to collagen in a small volume of abuffer. Any conventional buffer is useful, including e.g., DMEM. Thebuffer may be selected from a variety of buffers known to those of skillin the art to be used in the compositions of the invention and include,without limitation, phosphate buffered saline (PBS) or isotonic saline,such as ISOTON®II diluent, U.S. Pat. No. 3,962,125, [Beckman Coulter,Inc., Miami, Fla.], Tris buffer, the organic bufferN-(2-Acetamido)-2-iminodiacetic acid (ADA), or Pyrophosphate buffer orcombinations thereof. Also useful are acetate buffers, succinatebuffers, maleate buffers, citrate buffers, imidazole buffers, carbonatebuffers, MES buffer, MOPS buffer, and HEPES buffer, among many that maybe readily selected by one of skill in the art. Still other buffers suchas the Good buffers identified in Good, N. E. et al. 1966 Biochemistry5, 467 and Good, N. E., and Izawa, S. 1972 Methods Enzymol. 24, 53 maybe utilized depending upon the functional requirements of theformulation as determined by one skilled in the art.

Depending upon whether the cells embedded in the collagen have beenmanipulated to overexpress desired factors, the artificial stromalreconstruct is thereafter maintained in medium which may be completemedia, a minimal (or base) media, or base media supplemented withcertain essential and optional growth factors, as desired as discussedbelow.

In yet another embodiment, the artificial stroma is overlaid or coatedwith an extracellular matrix or matrix protein prior. In one embodiment,the fibroblast and/or smooth muscle cell-embedded collagen is treatedwith the matrix protein Laminin-2 α2β1γ1. In another embodiment thefibroblast and/or smooth muscle cell-embedded collagen is treated withthe matrix protein Laminin-1 α2β1γ1. In yet another embodiment, thefibroblast and/or smooth muscle cell-embedded collagen is treated with acombination of such matrix proteins. One commercially availablecombination is Matrigel® gel matrix (Collaborative Research, Bedford,Mass.), which contains Laminin-1 and other matrix proteins, such ascollagen IV and nitrogen. Preferably, the fibroblast and/or smoothmuscle cell-embedded collagen is treated with the matrix protein priorto seeding the epithelial cells.

Laminin-1 induces the polarization (differentiation) of carcinomaderived cells and normal cells. Laminin-2 increases the proliferationrate of epithelial cells in the organotypic culture. Generally, thefibroblast-embedded collagen is treated by addition of about 20 μg/ml oflaminin to the growth media in which the artificial stroma ismaintained. The artificial stroma is maintained in the laminin-enrichedmedia for about an hour, washed with base media, and placed in completemedia or base media enriched in factors in accordance with theinvention, with or without added laminin. If laminin is added, it isadded to about 10 μg/ml.

Matrix proteins, such as Laminin, from any source can be used to coatthe fibroblast-embedded collagen. Several commercially available matrixproteins include, without limitation, laminin 2 α2β1γ1 (human Laminin,Life Technologies, Rockville, Md.), laminin 1- α2β1γ1 (Sigma), mouselaminin 1 (Life Technologies), and Matrigel® matrix (CollaborativeResearch, Bedford, Mass.) (Burgeson, R E. et al., 1994 Matrix Biol.,14:209–211 and Page, K C. et al., 1990 Biol. Reprod., 43:659–664).

B. The Epithelial Cells

In the organotypic culture of this invention, epithelial cells areoverlaid or seeded on the surface of the artificial stroma. In oneembodiment, human intestinal cells are used. In another embodiment humancolonic or large intestinal epithelial cells are used. In anotherembodiment, human small intestinal epithelial cells are used. In oneembodiment the epithelial cells are adult human colon epithelial cells.In one embodiment, the epithelial cells are fetal human colon epithelialcells. In another embodiment, the epithelial cells are adult human smallintestinal epithelial cells. In another embodiment, the epithelial cellsare fetal human small intestinal epithelial cells. In still anotherembodiment, the epithelial cells are human epithelial stem cells. Suchstem cells may be obtained from human bone marrow. Such stem cells maybe embryonic stem cells.

The epithelial cells can be newly isolated from the above sources. Amethod of isolation of such cells is described in the Examples. See alsoRogler et al. 1998 Lab. Invest. 78: 889–900. Known alternative andmodified methods extant in the art can be used to isolate the humancolonic or small intestine epithelial cells. Alternatively, theepithelial cells can be derived from an established epithelial cellline, such as an adenoma or carcinoma cell line. Suitable cell lines maybe obtained from commercial or institutional laboratories andfacilities, such as the American Type Cell Culture, Manassass, Va.

In another embodiment, the epithelial cells for overlaying or seedingthe artificial stroma to form the organotypic culture of this inventionare genetically engineered to permit the cells to overexpress one ormore factors that are desirable for growth and maintenance of theculture. The techniques, vectors and factors useful for the generationof such genetically engineered epithelial cells are described in detailbelow, under the heading “Manipulation of Cells of the OrganotypicCulture”.

C. Optional Endothelial Cell Layer

In yet another embodiment, a layer of endothelial cells in a suitablemedium may be provided to underlie the artificial stroma and permit theorganotypic culture to become vascularized. These endothelial cells forma capillary network induced by the fibroblasts in the artificial stromaand infiltrate the collagen/fibroblast stroma to form microvessels. Sucha system is analogous to that described for a vascularized skinreconstruct in PCT Patent Publication No. WO02/30443, published on Apr.18, 2002 and incorporated by reference herein.

In one embodiment, human endothelial cells are used. In anotherembodiment, the endothelial cells are adult human endothelial cells. Inanother embodiment, the endothelial cells are fetal human endothelialcells. In still another embodiment, the endothelial cells are humanendothelial stem cells. Such stem cells may be obtained from human bonemarrow. Such stem cells may be embryonic stem cells.

The endothelial cells can be newly isolated from the above sources.Known methods extant in the art can be used to isolate the human colonicor small intestine endothelial cells. Alternatively, the endothelialcells can be derived from an established endothelial cell line. Suitablecell lines may be obtained from commercial or institutional laboratoriesand facilities, such as the American Type Cell Culture, Manassass, Va.

In another embodiment, the endothelial cells for underlaying theartificial stroma to form a vascularized organotypic culture of thisinvention are genetically engineered to permit the cells to overexpressone or more factors that are desirable for growth and maintenance of theculture. The techniques, vectors and factors useful for the generationof such genetically engineered epithelial cells are described in detailbelow, under the heading “Manipulation of Cells of the OrganotypicCulture”.

D. Manipulation of Cells of the Organotypic Culture

In certain embodiments of the present invention, the fibroblast oroptional smooth muscle cells present in the artificial stroma, theepithelial cells overlaying the stroma, and the optional endothelialcells may be engineered to express or overexpress a desirable factor orprotein to provide the organotypic culture with nutrients suitable forgrowth, proliferation, differentiation, and long-term survival. Includedamong these proteins are one or more of the factors which are otherwiseadded to the base media or growth media, as discussed below to assemblevarious embodiments of the organotypic intestinal culture of thisinvention.

In one embodiment of the organotypic culture, at least a portion of thefibroblasts used for admixture with collagen to fomn the artificialstroma are infected or transfected, prior to admixture with thecollagen, with a recombinant vector comprising a DNA sequence encoding aselected growth factor, under the control of regulatory sequencescapable of expressing that factor in the fibroblast. In one embodimentthe selected growth factor is a growth factor that binds the IGF-1receptor, such as insulin or IGF-1. In another embodiment the growthfactor is one that binds the EGF receptor, such as EGF or TGF-alpha. Instill another embodiment the growth factor is one that binds the LIFreceptor, such as LIF. In still another embodiment, the growth factorbinds the hepatocyte growth factor (HGF) receptor, such as HGF. TGF-β3is a factor that could be expressed to supply a factor alternative toHGF. In still another embodiment the growth factor is a growth factorthat binds the stem cell factor (SCF) receptor, e.g., SCF. In yetanother embodiment, the factor is a growth factor that binds theendothelin-3 (ET-3) receptor, such as ET-3. In another embodiment, thefibroblast is engineered to overexpress a growth factor that binds theplatelet derived growth factor (PDGF) receptor, e.g. PDGF. In stillanother embodiment, the fibroblast is engineered to express a matrixprotein, such as Laminin-1 or Laminin-2.

In an alternative or additional embodiment of an organotypic culture ofthis invention, at least a portion of the epithelial cells used forseeding the artificial stroma are infected or transfected before seedingwith a recombinant vector comprising a DNA sequence encoding a selectedgrowth factor, under the control of regulatory sequences capable ofexpressing said factor in the epithelial cell. In one such embodiment,the epithelial cell expresses a growth factor that binds the IGF-1receptor. In another embodiment, the epithelial cell overexpresses agrowth factor that binds the EGF receptor. In still another embodiment,the epithelial cell overexpresses a growth factor that binds the SCFreceptor. In yet another embodiment, the epithelial cell overexpresses agrowth factor that binds the autocrine motility factor (AMF) receptor.In still another embodiment, the epithelial cells overexpresses a growthfactor that binds the endothelin receptor A or the endothelin receptorB.

In still alternative embodiments in which smooth muscle cells are alsoembedded in the artificial stroma, the smooth muscle cells may also beengineered to express one or more desired growth factors or matrixproteins, including any one of the growth factors or proteins previouslyidentified above. In another embodiment of the organotypic culture, inwhich a layer of endothelial cells are employed to underlie thefibroblast-embedded artificial stroma, at least a portion of suchendothelial cells are infected with a recombinant vector comprising aDNA sequence encoding a selected growth factor or matrix protein, underthe control of regulatory sequences capable of expressing said factorprotein in the endothelial cells. One of skill in the art may select asuitable growth factor or protein for expression, if desired.

The components for the transfection or infection of one or more of theabove cell types in the culture are readily available in the art. Forexample, polynucleotide sequences encoding the proteins and growthfactors identified above, and plasmid and vector constructs containingthese sequences for expression in mammalian cells are known. Forexample, these sequences are available from GenBank or readilyaccessible scientific publications, or even commercially available. See,for example, LIF mRNA sequence (GenBank Accession No. XM009915); ET-3mRNA sequence (GeneBank Accession No. XM009583); and TGFP-3 mRNAsequence (GeneBank Accession No. J0324), and associated publications,thereof. One of skill in the art, e.g., in molecular biology, canreadily isolate, obtain, and manipulate these and other such sequencesfor expression, preferably regulated (inducible) expression of thosegenes.

For example, these sequences of the desired growth factors or proteins,useful fragments thereof, and modifications thereto may be constructedrecombinantly using conventional molecular biology techniques,site-directed mutagenesis, genetic engineering or PCR, and the like byutilizing the information provided herein. For example, methods forproducing the above-identified modifications of the sequences, includemutagenesis of certain nucleotides and/or insertion or deletion ofnucleotides, or codons, thereby effecting the polypeptide sequence byinsertion or deletion of, e.g., non-natural amino acids, are known andmay be selected by one of skill in the art. See, e.g., Sambrook et al.,Molecular Cloning. A Laboratory Manual., 2d Edit., Cold Spring HarborLaboratory, New York (1989); Ausubel et al. (1997), Current Protocols inMolecular Biology, John Wiley & Sons, New York).

Briefly described, a recombinant molecule or vector is constructed inwhich the polynucleotide sequence encoding the selected protein isoperably linked to a heterologous expression control sequence permittingexpression of the protein in the desired mammalian cells. Numerous typesof appropriate expression vectors and vector components suitable for usein this invention are known in the art.

Exemplary vectors and vector components, including selected constitutiveand inducible promoters, are readily available from a variety ofacademic and commercial sources, and include, e.g., adeno-associatedvirus (International patent application No. PCT/US91/03440), adenovirusvectors (M. Kay et al, 1994 Proc. Natl. Acad. Sci. USA, 91:2353 (1994);S. Ishibashi et al, 1993 Clin. Invest., 92:883 (1993)), or other viralvectors, e.g., various poxviruses, vaccinia, etc. In one embodiment ofthis invention, the desired vectors for use in infecting the cells ofthe organotypic culture are recombinant adenovirus vectors, includingsuch vectors deficient in the E1 gene and partially defective in the E3gene, such as those described in International Patent Publication No. WO98/39055, published Sep. 11, 1998 and incorporated herein by reference.Other known adenovirus vectors of the art may be similarly useful.

Exemplary regulatory sequences, including suitable promoters, may beselected for high level constitutive expression of the selected factoror protein in the cell of the organotypic culture of this invention,including, without limitation, the cytomegalovirus (CMV) promoter, theSV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter. Inducible promoters, regulated by exogenously suppliedcompounds, are also useful and include, without limitation, thezinc-inducible sheep metallothionine (MT) promoter, the dexamethasone(Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7polymerase promoter system; the tetracycline-repressible ortet-inducible systems. Other types of inducible promoters which may beuseful in this context are those which are regulated by a specificphysiological state, e.g., temperature, acute phase, a particulardifferentiation state of the cell, or in replicating cells only. Thenative promoter for the growth factor may be used and is when expressionof the transgene must be regulated temporally or developmentally, or ina tissue-specific manner. Tissue-specific promoters may also be used inthese vectors. Other native expression control elements, such asenhancer elements, polyadenylation sites or Kozak consensus sequencesmay also be used to mimic the native expression.

Thereafter, methods for ex vivo transduction, infection, or transfectionof such vectors in the cells of the present invention also are wellknown. See, e.g., Nesbit et al., 1999 Oncogene 18: 6469–76; Phaneuf etal. 2000 Mol. Med., 6: 96–103; and Satayamoorthy et al. 1997 CancerRes., 57: 873–6 (1997); International Patent Publication No. WO01/40455, published Jun. 7, 2001; International Patent Publication No.WO96/13597, published May 9, 1996, among others and the references citedabove.

The preparation or synthesis of the polynucleotide sequences andrecombinant vectors disclosed herein, as well as the components andtechniques useful for preparing same are well within the ability of theperson having ordinary skill in the art using available material. Theparticular selection of the vector, vector components, assembly methodsand transfection/infection methods used to generate the recombinantcells useful in this embodiment of the present invention are not alimitation of this invention. One of skill in the art may make aselection among these methods and components without departing from thescope of this invention and using the guidance provided by thisapplication.

E. Medium and Growth Factors

As discussed above, various media may be employed in the production ofthe components of the organotypic culture and to achieve the desiredproliferation, differentiation and survival of the culture.

For use in maintaining the artificial stroma prior to addition of theepithelial cells, a base media containing only the minimum nutrients isuseful. A suitable base medium can include MCDB 201 medium (Difco), L15medium (Sigma), DMEM with 10% fetal calf serum (FCS), among others.Other base media are known in the art. Optionally, such base media caninclude transferrin and serum components, such as FCS or antibiotics.One specific embodiment of a base medium useful for maintaining theartificial stroma contains 4 parts MCDB 201 medium, 1 part L15 medium(Sigma), 5 μg/ml transferrin (Sigma), and the antibiotics, streptomycinand gentomycin, 50 μg/ml, each.

In embodiments of this invention in which the cells in the organotypicculture are not engineered to overexpress certain growth factors, oronly engineered to express one or less than all of the necessaryfactors, the medium used in the organotypic culture can supply thefactors exogenously. Along with the release of fibroblast-derived growthfactors, colonic epithelial cells in the organotypic culture requireexogenous growth factors in culture medium for survival, especiallyduring the first 3 days of culture. During this time, thecollagen-constricting fibroblasts have not yet reached a homeostaticbalance. Besides growth, migration of the colonic cells appears to becritical for homeostasis of the colonic epithelium. Such growth factorsare commercially available from a variety of sources, identified in theExamples below.

A minimally supporting base medium for maintaining the organotypicculture of this invention further includes a growth factor that binds tothe epidermal growth factor (EGF) receptor, a growth factor that bindsto the insulin-like growth factor-1 (IGF-1) receptor, and a growthfactor that binds to the leukemia inhibitory factor (LIF) receptor.Growth factors that bind the IGF-1 receptor include insulin and IGF-1and are survival factors. Growth factors that bind the EGF receptorinclude EGF and tumor growth factor-alpha (TGF-alpha). EGF can stimulateintestinal epithelial cells in vivo and enhance colonogenic growth invivo (Chailler, P. and Menard, D., 1999 Front. Biosci., 4:D87–101).Among growth factors that bind the LIF receptor is LIF. LIF was the mostsignificant factor for proliferation of eukaryocytes while inhibitingdifferentiation. This factor can be secreted by colorectal carcinomacells to stimulate their proliferation (Kamohara, H. et al 1994 ResCommun Mol Pathol Pharmacol 85, 131–140) by colorectal carcinoma cellsto stimulate their proliferation (Guimbaud, R. et al, 1998 Eur CytokineNetw 9, 607–612; and Bellone, G. et al, 1997 Cell. Physiol., 172, 1–11)by binding to its receptor expressed by colonic epithelial cells(Rockman, S. P et al, 2001 J. Gastroenterol. Hepatol. 16, 991–1000) bybinding to its receptor expressed by colonic epithelial cells (Rockman,S. P. et al, 2001 J. Gastroenterol Hepatol 16, 991–1000). LIF activitywas dependent on collagen-embedded fibroblasts. In the presence of theessential growth and survival factors insulin and epidermal growthfactor (EGF), leukemia inhibitory factor (LIF) had the most profoundeffect on stimulation of proliferation of the epithelial cells in theorganotypic culture, while preventing differentiation, indicating thatLIF is critical for maintaining the phenotype of colonic crypt cells.

In contrast to the minimally supporting medium for the organotypicculture, a complete growth medium generally consists of the base mediumsupplemented with a variety of growth factors in addition to thoseidentified in the minimally supporting medium. In complete growthmedium, the growth factors induce a balance between proliferation anddifferentiation. Among such additional growth factors includes, withoutlimitation, a protein that binds the human basic fibroblast growthfactor (bFGF) receptor, a protein that binds the endothelin-3 (ET-3)receptor, a protein that binds the endothelin receptor A or B, a proteinthat binds the hepatocyte growth factor (HGF) receptor, a protein thatbinds the stem cell factor (SCF) receptor, a protein that binds theautocrine motility factor (AMF) receptor, and a protein that binds theplatelet derived growth factor (PDGF) receptor. Optionally, such mediacan include transferrin and serum components, such as fetal calf serum(FCS). See, e.g., the medium described in Nesbit M., et al. 1999Oncogene 18:6469–6476.

For example, one embodiment of a suitable growth media for theorganotypic cultures includes EGF, LIF and insulin, and at least one ofSCF and ET-3. As demonstrated in the Examples, LIF was synergisticallyactive with SCF and ET-3, which show little activity on their own. LIFalso synergizes with HGF, which on its own induces a flat, migratoryepithelial phenotype (Nusrat A. et al., 1994 Clin. Invest. 93:2056–2065). LIF may also have an effect on goblet cells which form inthe organotypic construct of this invention. Goblet cells are terminallydifferentiated due to their high levels of mucin production. As shown inthe examples below, the inventors observed that 33% of goblet cells inthe organotypic culture incorporated BrdU during a 10-day cultureperiod, suggesting that they developed through differentiation ofproliferating enterocytes. LIF does not induce differentiation of cellsto the neuroendocrine phenotype, which steadily declined in the culture,suggesting that other factors are needed for this cell type. LIF wascritical for proliferation of enterocytes and inhibited expression ofthe differentiation marker carbonic anhydrase II. In the presence ofLIF, the number of goblet cells remained stable, whereas enteroendocrinecell number declined. LIF stimulation of cultures remained dependent onthe presence of fibroblasts in the matrix. In the absence of othergrowth factors, LIF induced formation of disorganized structures ofstratified and semi-stratified cells, suggesting that the homeostaticbalance in the normal human colon requires cooperation withdifferentiation-inducing factors.

In still another medium used in the examples, a complete growth mediumcontained a base media and insulin, transferrin, EGF, ET-3, HGF, LIF,SCF, AMF, and FCS, which combined to stimulate growth, migration anddifferentiation of the colonic cells. Only cultures in HGF-containingmedium were able to cover the entire collagen matrix, consistent withevidence that this growth factor provides migratory stimulation.Although EGF can also stimulate migration, it alone was not sufficientto initiate it in the organotypic reconstruct of the invention.

In combination with other factors in the medium, LIF was one of the mostimportant mitogens and morphogens. In the presence of LIF and EGF only,colonic cells formed a polarized monolayer of highly cylindrical cells.Proliferation and cell-type specific morphology were further enhancedand altered, respectively, by the combination of SCF, ET-3, EGF, andinsulin. Under these conditions, epithelia formed highly disorganizedstructures of stratified and pseudostratified cells. Goblet cells lostpolarity and deposited mucus towards the basement membrane. The numberof goblet cells decreased in LIF-containing media, most likely becauseLIF can inhibit differentiation. Dysplastic morphology and decreasednumber of goblet cells are early indications of premalignancy in thehuman colon (Wargovich, M J. et al. 1983 J. Natl. Cancer Inst.,71:125–131 and Archer, M C. et al. 1992 Environ. Health Perspect.,98:195–197). In complete medium, other not yet identified growth factorsmust have induced a balance between proliferation and differentiation.

Still other specific media for use in maintaining the organotypiccultures of this invention are described with specificity in theExamples below.

II. Methods for Preparing an Organotypic Culture of this Invention

In general, the method of preparing an organotypic culture of theinvention comprises the primary steps of assembling the artificialstroma by mixing collagen and fibroblasts as described in detail aboveand thereafter, seeding the artificial stroma with epithelial cells inthe presence of a growth factor that binds the IGF-1 receptor, a growthfactor that binds the EGF receptor, and a growth factor that binds theLIF receptor. As described above the growth factors may be providedexogenously by the media or may be provided by the use of recombinantlyengineered fibroblasts or epithelial cells that express one or more ofthese factors.

However, more complex organotypic cultures of the invention may beprepared by layering the artificial stroma over an endothelial celllayer, as described above. Additionally or alternatively, a matrixprotein, e.g., laminin may be used to coated the artificial stroma priorto the seeding of the epithelial cells. Still other embodiments areprovided by the use of smooth muscle cells or neural cell types added tothe artificial stroma. Still a further embodiment of an organotypicculture of this invention is provided by seeding a malignant cell on thesurface of the artificial stroma, such as a epithelial cancer cell. Bysuch addition of a cancer cell type on the surface of the artificialstroma, the organotypic culture can become a model of tumor formation ortumor-stroma interaction. Examples of readily available malignant celltypes are a tumor from a human patient, or established tumors in ananimal, or established malignant cell lines. In lieu of malignant cellseeding, a whole tumor could be implanted in a reconstruct. See, e.g.,Ochalek and Kleist, 1993 Clin. Lab. Anal., 7: 155–163.

In one embodiment for preparation of an organotypic culture of thisinvention, colonic epithelial cells or small intestine epithelial cellsin media are seeded or overlaid on the surface of the artificial stroma.In one embodiment of the method of generating the culture, the mediavolume is reduced from the time of overlaying and for about 1 to about1.5 hours, to allow the epithelial cells to attach to the artificialstroma. The seeding can occur at anytime after the artificial stroma isestablished. The seeding preferably occurs within 24 hours after theartificial stroma is assembled.

The resulting organotypic reconstruct is grown on a minimally supportingmedium, i.e., base media plus LIF, IGF-1 or insulin, and EGF orTGF-alpha, or various formulations of complete media, including the basemedium plus optionally any other factor(s) from among the growth factorsidentified above, transferrin or 1–2% FCS. The growth conditions (e.g.,temperature, oxygenation, resemble standard mammalian cell growthconditions. For example, the culture is incubated at 37° C. in 5% CO₂ ingrowth media which was changed daily for 7 days, then changed threetimes/week for the next 21 days.

The fibroblasts and optionally smooth muscle cells act to constrict thestromal reconstruct and allow epithelial cells to expand and cover theorganotypic culture. The close proximity of the fibroblasts toepithelial cells in the organotypic culture allows formation of amicroenvironment that closely mimics mucosal compartments in vivo. Thismicroenvironment includes the formation of the three types of cells thatnormally populate the intestinal epithelial wall, e.g., goblet cells,enteroendocrine cells and enterocytes. The inventors have maintained theepithelial cells and normal colonic homeostasis of cell growth anddifferentiation in the organotypic culture containingfibroblast-embedded artificial stroma in a complex growthfactor-supplemented medium for more than 1 month, and up to 40% of theentire cell population had proliferated during a 10-day incubationperiod.

Goblet cells proliferated or differentiated from proliferatingenterocytes, whereas enteroendocrine cells were only maintained withoutproliferation. Cooperative effects of growth factors were dependent onthe presence of fibroblasts. The migratory properties of colonicepithelia in the organotypic culture of this invention resembledintestinal wound healing with epithelial restitution (Dignass, A. U.,2001 Inflamm. Bowel Dis., 7:68–77).

As described in the Examples below, in one embodiment of an organotypicculture of this invention, human fetal colonic epithelial cells wereisolated and seeded on a collagen type I matrix embedded with colonicfibroblasts. The epithelial cells rapidly spread from clusters andproliferated, and within 3 days, a columnar layer of polarizedepithelium surrounded the surface of the constricted collagen matrix.The polarized enterocytes developed brush borders, tight junctions anddesmosomes, and goblet and enteroendocrine cells were dispersedthroughout the epithelium. A balance of growth and differentiation wasmaintained for several weeks in the presence of collagen-embeddedfibroblasts and a complex mixture of growth factors.

In another embodiment, the artificial stroma containing fibroblasts andsmooth muscle cells constricted the collagen down to about 10% of itsoriginal volume, allowing the epithelial cells to migrate andproliferate around the entire collagen matrix, producing an organotypicculture of this invention.

As described in the Examples below, several embodiments of theorganotypic cultures of this invention were grown either in a completemedia, i.e. the base media supplemented with insulin, transferrin, EGF,ET-3, HGF, LIF, SCF, AMF, and only 1% FCS, which combined to stimulategrowth, migration and differentiation of the cells, or in a base mediasupplemented with 1% FCS, insulin, and EGF (the minimal media) andsubsets of the other factors, which identified some of the factors to becrucial for specific phenomena, i.e. cell migration, survival,proliferation and differentiation. In other embodiments, prior to theseeding of epithelial cells, the stromal reconstruct is incubated in themedia further including laminin-1 and/or laminin-2, as described above.

This organotypic culture of normal human colon cells provides evidencethat microenvironmental factors regulate the proliferation anddifferentiation of these cells. Both the presence of stromal cells andgrowth factor supplementation in the medium were critical. When theintestinal organotypic reconstruct was grown in the complete media, theepithelium quickly covered all free surfaces and maintained a polarizedmonolayer with a balance of proliferation and apoptosis. There wasdifferentiation into all major epithelial cell types, includingenterocytes, goblet cells, and neurosecretory cells. In the intestinalreconstruct of the present invention, the mesenchymal (stromal) cellswere observed to actively migrated and accumulated beneath theepithelium and aligned with the epithelium cells. This is likely due toattractants released by the colonic cells or to the trapping of randomlymigrating fibroblasts at the epithelial interface and the developingbasement membrane. During contraction of the collagen gel, the majorityof the mesenchymal cells expressed α-smooth muscle actin (α-SMA),indicating differentiation to myofibroblasts., as also observed infibroblasts exposed to TGF-β (Berking, C., et al, 2001 Cancer Res.,61:8306–8316). Myofibroblasts, i.e., mature and differentiatedfibroblasts, and mature smooth muscle cells are known to produce andexcrete growth factors including HGF, bFGF, IGF, and KGF (Nusrat A. etal., 1994 J. Clin. Invest. 93: 2056–2065 and Powell, D. W., et al, 1999Am. J. Physiol., 277:C1–9). Fibroblasts directly subjacent to theepithelium continued to express A-SMA for prolonged periods, as is seenin situ, suggesting that epithelial-derived signals induce amyofibroblast phenotype (Sappino, A P. et al., 1989 Virchows Arch. A.Pathol. Anat. Histopathol. 415:551–557). The contribution of mesenchymalcells to epithelial cell growth and differentiation is thus two-fold: 1)production of soluble growth factors; and 2) production of matrixproteins that function as basement membrane components. Theseorganotypic reconstructs of the invention are viable for about onemonth.

Besides growth, migration of the epithelial cells appears to beimportant for homeostasis of the intestinal reconstruct. Only incultures in HGF-containing medium were epithelium cells able to coverthe entire collagen matrix, consistent with evidence that this growthfactor provides migratory stimulation. EGF also has been reported tostimulate migration (Basson, M. D. et al, 1992 J Clin Invest 90, 15–23).

Specific morphologies and states of organization were observed by growthin the base media plus 1% FCS and sub-combinations of the other factors.LIF was one of the most critical mitogens and morphogens. In thepresence of LIF and EGF only, epithelial cells formed a polarizedmono-layer of highly cylindrical cells. Epithelia showed disorganizedand undifferentiated growth in media containing LIF, EGF and insulin.Proliferation and cell-type specific morphology were further enhancedand altered, respectively, by the combination of SCF, ET-3, EGF, andinsulin. Under these conditions, epithelia formed highly disorganizedstructures of stratified and pseudo-stratified cells. Goblet cells lostpolarity and deposited mucus towards the basement membrane.

The number of goblet cells decreased in LIF-containing media, mostlikely because LIF can inhibit differentiation. The dysplasticmorphology and decreased number of goblet cells are early indications ofpre-malignancy in the human colon (Wargovich M J. et al., 1983 J. Natl.Cancer Inst., 71:125–131 and Archer, M C. et al., 1992 Environ. HealthPerspect., 98:195–197). In complete medium, other growth factors induceda balance between proliferation and differentiation.

In other embodiments of the organotypic culture of this invention, whenthe stromal matrix layer was coated with laminin 1, differentiation ofthe colonic epithelial cells was induced. By contrast, when the stromalmatrix layer was coated with laminin 2, the cells were slower topolarize and proliferation of the cells was observed.

The intestinal reconstruct of the invention has multiple uses asdiscussed below. For specific uses, it is advantageous to employ growthconditions, components and media, as discussed, which produce anorganotypic culture with the desired characteristics. It should furtherbe noted that the overall size of the reconstruct can be varied. Thereconstruct can be up to about 2 cm in length and up to about 7 mmthick. If desirable, the constriction level of the reconstruct can bemanipulated by the amount of fibroblast introduced and the level ofgrowth factors in the media. The standard conditions described in theexamples lead to constriction of the stromal reconstruct up to about 10%of its original volume. Generally, a greater constriction results in asmaller but tougher, more easy to manipulate reconstruct, but lessconstriction leads to a larger and more elastic reconstruct. Reductionsto 5% to 20% of the original volume are possible. Still otherembodiments of the organotypic cultures of this invention are discussedbelow in the Examples.

III. Methods of Use of the Organotypic Culture

As discussed above, the organotypic intestinal reconstructs of theinvention have multiple uses. This new reconstruction model of thenormal human colon is useful in a method for identifying factorsinvolved in the homeostatic balance of normal colonic epithelium and inits dysregulation. The results of the construction of the cultures invarious supplemented media provide evidence that an imbalance of growthfactors in colon epithelia contributes to transformation of theepithelium. This information thus allows for use of organotypicreconstructs in accordance with the invention for identification ofcompounds, molecules and growth factors or any other natural orsynthetic agents that control or affect cell differentiation,proliferation, migration, malignancy, metabolism, transport, homeastasyand the like. Because they are relatively easy to generate in areproducible manner, the organotypic cultures can be used advantageouslyfor a number of purposes including hormone and growth factor regulatoryinfluences as well as cell-cell and cell-matrix interactions. Otherpotential applications for the organotypic cultures are numerous andinclude the analysis of drug and nutrient transport and metabolism andthe study of microorganism intestinal epithelial cell interactions.

For example, screening assays are useful to identify therapeutic agents,or to determine if agents are carcinogenic, or to allow study of drugcandidates in terms of their effect on the tissue and their absorptionin the tissue or localization in a type of cell or cellular compartment.A skilled artisan will readily appreciate that certain physiological ormorphological embodiments of the organotypic cultures are more suitablyadapted to specific purposes. The observations herein on the effect ongrowth, organization, polarization, generation of cell types, andmovement of the cell types of the reconstruct grown in the varioussubsets of factors added to the minimal media allows the skilled artisanto employ a particular reconstruct in accordance to the invention.

For example, drug absorption studies may preferably utilize theorganotypic culture containing small intestine epithelial cells asopposed to colonic epithelial cells, because absorption occurspredominantly in the small intestine. When evaluating an agent for itspotential negative effect on cell replication, it may be advantageous togrow the organotypic reconstruct in a combination of growth factorsand/or laminin-1 or laminin 2. The combination of factors is selected bythe person of skill in the art to enhance or inhibit cell proliferation,or enhance or inhibit cell differentiation, depending on the specificeffect expected of the drug candidate. Further for drug screening, theculture of the invention may be grown in base media plus EGF, insulinand optionally only 1% fetal calf serum (FCS), unlike the more typicalcomplex media or minimal media fortified by higher levels of FCS,typically 10%. Lower levels of FCS in media are desired for drugscreening because FCS may mask, inhibit, degrade or compete with theeffects of specific drug candidates.

The agents to be tested in the various assays described below can befrom any source. For example, natural or isolated factors, proteins,polypeptides or fragments, or combinations thereof, or syntheticversions thereof, chemical agents, synthetic molecules and the like, maybe assayed. Food ingredients may also be assays. Synthetic chemicals,biochemicals, or library of factors can be tested. The agent tested maybe labeled for convenience of detection and/or isolation. Labeling canbe by any method known in the art, example by conjugation labeling, byradiolabeling or by addition of an affinity tag sequence to the primarysequence (for example a His amino acid sequence to the end of a proteinsequence). Alternatively, an unlabeled factor can be detected after theassay, by immune assays, enzymatic assays, or metabolic assays. Theselection of the label, and the type of detectable assay (e.g.,immunoassays, enzymatic assays and the like) to further detect theagent's presence in the culture over time, may be readily selected byone of skill in the art.

Thus, in one embodiment, a method of in vitro screening of an agent canbe accomplished by contacting an organotypic culture of this inventionwith the agent in a vessel, and observing the effect of the agent uponthe culture. For example, in one such method, the agent is a drugcandidate, and screening includes determining the absorption rate of thedrug candidate by measuring and observing the movement of the drugcandidate through the epithelial cell layer at the top of the culture,optionally as a function of time.

In one embodiment, an assay that permits screening for absorption of adrug candidate employs the organotypic culture prepared with human smallintestinal cells, seeded on an artificial stroma coated with a matrixprotein, e.g., Laminin-1 or Laminin-2. Preferably, the reconstruct wouldbe grown in complete media. Depending on the identity of the drugcandidate, it may be labeled with a conventional detectable label.Alternatively, an enzymatic assay or immunoassay is available to detectthe drug candidate. When the epithelial cells are still young but welldeveloped on the culture (about 5 days after seeding), the drugcandidate is spotted on a small surface area on top of the reconstruct.The culture is observed. The presence of the drug appearing at thebottom of the vessel in which the reconstruct is grown or in cellsisolated from the reconstruct at a site away from the application site,as detected by the label, enzymatic assay or immunoassay, is indicativeof absorption. In an alternative embodiment, the drug candidate isplaced at the growth vessel at the bottom of the culture and itsdetection in epithelial cells at the top of the culture is similarlymonitored. In still another embodiment of a screening assay, the rate ofabsorption is determined in time controlled experiments.

Another screening assay involves screening the agent for toxicity tohuman intestinal tissue. In this embodiment of the method, the observingstep involves observing the effects of the agent on the morphology andlife span of the epithelial cells, whereby an agent which reduces thelife span of the epithelial cells or has a negative impact on themorphology of the epithelial cells is toxic. Similarly, the screeningmethods of this invention can include screening the agent for itseffects on hormone regulation in the culture or screening the agent forbinding for a receptor in the culture.

Still another use of the organotypic culture of this invention is in amethod for screening an agent for repairing effect on intestinalepithelial cell injury. According to this type of assay, the layer ofepithelial cells on an organotypic culture is disrupted. The site ofdisruption is contacted or exposed to the agent. Observing the effectsof the agent on the repair of the epithelial cell layer permits adetermination that an agent which promotes repair of the epithelial celllayer is capable of repairing the injury. In one embodiment, such anassay to screen for agents that can promote healing of a wound in acolonic/intestinal tissue employs an organotypic culture of thisinvention that was grown in the complete media and, preferably, has anartificial stromal layer that was coated with Laminin-1 and/orLaminin-2. When still young but well developed (about day 4), theorganotypic reconstruct would be wounded. The wound could be mademechanically (a scratch), or by a temporary/local exposure to an acid orto a base, or a virus, etc. The wounded reconstruct would be allowed torecover in the presence of a drug candidate. Proper controls may includea similarly constructed and damaged reconstruct left without treatment.Comparison of the rate of repair and the final repair condition of thetreated culture vs. the control culture would permit identification of asuitable therapeutic agent.

In another example, an assay is designed for screening for agents thatcan promote healing of a malignancy and destroy or inhibit growth ofmalignant cells in colonic/intestinal tissue. The organotypic cultureuseful in this invention would be the embodiment described above whichis additionally seeded on the surface of the artificial stroma with amalignant cell type, for example a cancerous or malignant tumor cell.Once the tumor cell is developed on the organotypic culture, the cultureis contacted with the agent. The effect of an agent or a drug candidateon the proliferation, growth or general morphology of the malignant celltype would be observed. Proper controls may include a similarlyconstructed and seeded or implanted reconstruct left without treatmentby the drug candidate.

Still another example of a screening assay of this invention involvesstudying the effect of an agent, for example an environmental factor ora food type, on the proliferation, differentiation or survival of cellsin the culture. For the study of differentiation, the embodiment of theorganotypic culture that may be used optionally contains the stromacoated with Laminin-1. If the effect on cell proliferation is to bestudied, the culture may be advantageously grown in a minimal medium(EGF, insulin, 2% FCS).

One of skill in the art will recognize other possible assays, includingassays that use multiple agents to determine their combined effects onthe organotypic culture, and the preferred embodiment of the organotypicculture to elucidate the specific question of the assay.

In another embodiment, the culture of this invention is useful in amethod for enhancing epithelial cell repair at an in vivo site ofintestinal or colonic injury. According to this method, at least one ofcollagen, fibroblasts, at least one growth factor that binds the IGF-1receptor, at least one growth factor that binds the EGF receptor, andLIF, and a combination thereof are delivered to the site of the injury.The delivery can include administering to the site of the injury atleast one recombinant vector comprising a polynucleotide moleculeencoding at least one of a growth factor that binds the IGF-1 receptor,a growth factor that binds the EGF receptor, a growth factor that bindsthe LIF receptor, or a combination thereof. The vector may be present ina transfected or infected fibroblast delivered to the site of theinjury. The vector may be present in transfected or infected intestinalor colonic epithelial cells delivered to the site of the injury. Themethods of delivery may be selected by the attending physician withregard to the nature of the injury, and the particular therapeuticcomposition being administered. However, it is anticipated that localadministration would be preferred. The amounts and dosages of suchvectors or organotypic components would also be selected by one of skillin the art.

Still another use of the culture of this invention is in treating anintestinal wound by placing an organotypic culture on the intestinalwound in a patient, i.e., using the culture as a tissue replacement in asurgical procedure. Preferably at least one cell present in saidorganotypic culture is allogeneic to the patient receiving treatment.This method is useful in treating wound is caused by, inter alia,chronic inflammation, Crohn's disease, ulcerative colitis, intestinalhemorrhage, hemorrhaging diarrhea, ulcers, or a malabsorption syndrome.Such wounds may also be the result of surgery, e.g., a post operativewound, or irradiation, e.g., a post irradiation treatment.

Typically the reconstruct used for treatment would be one that mostclosely resembles the in situ tissue. In that respect, a treatment wouldpreferably employ a reconstruct made by the seeding of small intestinecells. Preferably, the reconstruct would be grown on the complete media.In accordance with another embodiment, at least one of the cell typesused to construct the intestinal reconstruct is allogeneic, i.e.,derived from the patient who will receive treatment.

The following examples are provided to illustrate the invention and donot limit the scope thereof. One skilled in the art will appreciate thatalthough specific reagents and conditions are outlined in the followingexamples, modifications can be made which are clearly encompassed by thespirit and scope of the invention, which modifications do not involveundue experimentation on the part of the person of skill in the art.

EXAMPLE 1

Preparation of Organotypic Cultures

A. Cells

Human colonic fibroblasts were established from explants of colons from7- to 20-week-old fetuses of therapeutic or spontaneous abortions. Thecolonic explants were obtained through Advanced Bioscience Resources(Alameda, Calif.), after approval by the Institutional Review Board. Outof three fibroblast cell lines established from these explants, FFC331was used for most of these studies. The fibroblasts were cultured inDulbecco's modified minimum essential medium (DMEM, GIBCO BRL,Rockville, Md.) supplemented with 10% fetal calf serum (FCS, Cansera,Rexdale, Ontario, Canada) and antibiotics. Cultures were used up topassage 10.

Human smooth muscle cells (HIAS 119) were isolated from large vesselsand maintained in medium M199, supplemented with 10% FCS, 2 mML-glutamine, and 50 μg/ml of bovine hypothalamic extract (Sorger, T. etal. 1995 In Vitro Cell Dev. Biol. Anim., 31:671–683).

Human enteric epithelial cells were isolated by dissociation from thecolon specimens within 24 hr after surgery. The colon lumen was openedand washed in Hank's buffered salt solution (HBSS; Gibco BRL)supplemented with penicillin at 200 U/ml, streptomycin at 200 μg/ml(30-002-CL, Cellgro, Herndon, Va.) and gentamycin at 100 μg/ml(30-005-CR, Cellgro). After removal of the serosa, the remaining tissuewas incubated for 10 min in HBSS containing 20 mg/ml Mucomyse(N-acetyl-L-cystein; Sigma, St. Louis, Mo.), pH 7.2, to removesurface-bound mucin. After three washings, the tissue was dissociated at37° C. in HBSS without calcium and magnesium (GIBCO BRL), supplementedwith 1 mg/ml D-glucose (Sigma) and 1 mM ethylene diaminetetraacetic acid(EDTA; Sigma). After 10-minute incubation with occasional shaking,single cells and small cell clusters were removed and collected in a2-fold volume of base medium supplemented with 5% FCS. The procedure wasrepeated twice. All collected epithelia were pelleted at 800×g for 10minutes. Single cells and cell clusters were washed and then resuspendedin complete growth medium.

B. Media

Base medium consisted of 4 parts of MCDB 201 medium and 1 part of L15medium (Sigma), 5 μg/ml of transferrin (Sigma), and 50 g/ml ofstreptomycin and gentamycin, respectively. Complete growth mediumconsisted of base medium supplemented with recombinant human basicfibroblast growth factor (bFGF) at 10 ng/ml, human recombinant epidermalgrowth factor (EGF) at 10 ng/ml (Sigma), insulin at 5 pg/ml (Sigma),endothelin-3 (ET-3; Peninsula Labs, San Carlos, Calif.) at 264 ng/ml,hepatocyte growth factor (HGF; R&D Systems, Minneapolis, NIN) at 30ng/ml, leukemia inhibitory factor (LIF; R&D Systems) at 0.2 ng/ml, stemcell factor (SCF; Sigma) at 30 ng/ml, autocrine motility factor (AMF,Sigma) at 35 ng/ml, and 1% FCS (Nesbit M., et al. 1999 Oncogene18:6469–6476).

To determine the role of growth factors on proliferation of colonicepithelial cells, this growth medium was modified by changingcombinations of growth factors and deleting serum. Keratinocyte growthfactor (KGF, R&D Systems) at 20 ng/ml was also tested as part of 23growth factor combinations.

C. Preparation of the Organotypic Culture.

In this embodiment and others in which smooth muscle cells areintroduced into the artificial stroma, the artificial stroma isgenerally produced by mixing fibroblasts and smooth muscle cells in a10:1 ratio within the collagen. Specifically, collagen gels withembedded colonic fibroblasts and smooth muscle cells (also referred toas artificial stroma or stromal reconstructs) were obtained bysuspending bovine tendon acid-extracted collagen (Organogenesis, Canton,Mass.) to a final concentration of 0.9 to 1.1 mg/ml in DMEM supplementedwith Vitamin C (Sigma) at 50 μM/L, L-glutamine (GIBCO-BRL) at 1.66 mMand 1% FCS. The suspension was neutralized, pH 7.2, with 7.5% sodiumbicarbonate. Before the collagen gel hardened, colonic fibroblasts(2×10⁵/ml) and smooth muscle cells (2×10⁴/ml) were added. Chamberedslides, Lab-Tek 8 or Lab-Tek 16 wells (Nalgene Nunc International,Rochester, N.Y.) were filled with 0.3 and 0.2 ml of the collagen/cellmixture, respectively.

Single epithelial cells and small cell clusters were freshly isolatedfrom the mucosa of fetal colon using mild EDTA treatment. Aftersolidification of the artificial stroma, these freshly isolated colonicepithelial cells in medium were seeded on the surface of the stroma toestablish the organotypic culture. Specifically, the cultures wereprepared by seeding epithelial cells in 0.4 ml of complete growthmedium. After seeding, epithelia were incubated for 60–90 minutes in alow volume of medium (50–100 μl) to enhance attachment. The base mediumof MCDB201/L15 with transferrin and antibiotics was supplemented withEGF, Insulin, HGF, bFGF, ET-3, LIF, SCF, AMF, and 1% FCS. The coloniccells began attaching within 60 minutes unless they were surrounded bymucin.

D. Characterization of Organotypic Cultures.

For evaluation of proliferation, cell proliferation labeling reagent(Amersham, Pharmacia Biotech, Inc., Piscataway, N.J.) was added to themedium according to the manufacturer's instructions. Harvested cultureswere fixed in 1.5% paraformaldehyde followed by characterization,embedded in paraffin for sectioning and processed for microscopy. Forelectron microscopy, they were fixed in 4% glutaraldehyde.

For histochemistry and immunohistochemistry, 5 μm thick sections werecut from the paraffin beds and stained with hematoxylin and eosin (H&E),PAS, or Alcian Blue using standard procedures. Antibodies for stainingwere against bromodeoxyuridine (BrdU, Amersham Pharmacia Biotech, Inc,Piscataway, N.J.), the proliferation marker Ki67 (DAKO, Carpinteria,Calif.), and α-SMA (Sigma). Immunohistochemistry was performed usingstandard protocols.

Total relative cell numbers of a specific type were counted as anaverage of all nuclei counts from a minimum of 3 randomly selectedcross-sections of a reconstruct. Goblet cells were counted based ontheir typical morphology and positive Alcian staining in at least 3randomly selected slides. Proliferation rates in % were established bycounting the number of BrdU or Ki-67-positive nuclei per totalepithelial cell nuclei of section. Apoptotic cells were counted inH&E-stained sections (Vagunda, V. et al. 2000 Anal. Quant. Cytol.Histol., 22:307–310). Apoptotic bodies and nuclei were determinedaccording to the size and numbers of the fragments. Apoptosis wascalculated as percentage of total nuclei per section (Kerr, J F. et al.1972 Br. J. Cancer, 26:239–257)

E. Results of Characterizations

In the first embodiment of the organotypic culture described above,spreading of cells from clusters was seen after 12 hours. Thetissue-like/structure was formalin-fixed 12 hours after seeding, andthen sectioned and stained with hematoxilin and eosin (H&E). A clusterof epithelial cells attached in the middle of the culture, and flatcells began covering the free surface. After 24 hours, the cells hadmigrated over the entire matrix and formed a flat monolayer. At thistime, the collagen began to shrink due to contraction by the fibroblastsand smooth muscle cells. Maximum shrinkage was reached by day 3. Thereconstructs at this point were 3 mm long and about 1.5 mm thick. Day 4after seeding an epithelial monolayer of polarized colonic cells hasformed. Goblet cells are dispersed throughout the epithelial layer.Fibroblasts and smooth muscle cells are found throughout the organotypicculture. The artificial stroma has constricted to approximately 10% ofits original volume. With contraction of the collagen matrix, a changeof the epithelial phenotype occurred, from flat on day 1 to cuboidal andcolumnar on day 4.

By day 10 after seeding, a continuous, well-polarized epithelial layerhad covered all sides of the organotypic culture. Fibroblasts and smoothmuscle cells remained dispersed throughout the collagen, but somefibroblasts migrated towards the epithelial layer and closely alignedbelow it. Mucin-producing goblet cells, identified by their morphologyand Alcian blue positive staining, were dispersed throughout theepithelial cell layer. In complete growth medium, the percentage ofgoblet cells remained stable until day 10. By day 20, the monolayer ofepithelial cells remained intact but the internuclear distancesincreased. Goblet cells were still present by day 28, when the totalcell number had decreased.

Electron micrographs of the colon epithelium in organotypic culturedemonstrated formation of well-developed microvilli on the apicalsurface of the epithelial cells. The upper lateral margins of theepithelial cells were connected by tight junction complexes.Intercellular adhesion complexes were formed by desmosomes andinterdigitating folds. Brush borders and tight junctions were alsoidentified in mucin-producing goblet cells. Neuroendocrine cells werefilled with neurosecretory vesicles and lysosomes. Between theepithelial and mesenchymal layers, an immature basement membranedeveloped.

Immunohistochemistry of the organotypic culture 10 days after seeding bystaining for the presence of α-SMA showed positive fibroblasts,identified by their rough endoplasmic reticulum, tightly alignedsubjacent to the basal side of the epithelial cells. These fibroblastsresembled intestinal sub-epithelial myofibroblasts, regardless ofwhether a mixture of fibroblasts and smooth muscle cells or onlyfibroblasts were used for the artificial stroma. Only cells adjacent tothe epithelium were positive for α-SMA. In a section of the fetal colon,similarly stained for α-SMA, an alignment of stained mesenchymal cellswere observed beneath the epithelial layer of the myofibroblasts belowthe crypts.

Two to 3% of epithelial cells on the stromal reconstructs stainedpositive for the proliferation markers Ki67 and BrdU on day 10, and 4.5%of the epithelial cells visible in the sections appeared apoptotic.

In control cultures, colonic epithelial cells were seeded on plasticdishes coated with collagens I and III or Matrigel matrix, definedabove. The colonic epithelial cells remained flat and died within 8days. In co-cultures of fibroblasts, smooth muscle cells, and epithelialcells, colonic epithelial cells were overgrown by fibroblasts and smoothmuscle cells and died after a few days.

EXAMPLE 2

Growth Factor Modulation of the Epithelial Phenotype

To define the role of critical growth factors supporting growth anddifferentiation of the enteric epithelial cells, an organotypic culture,such as that described in Example 1, was grown in medium in which thenumber of supplements was reduced.

In one experiment the base medium (MCDB 201/L15 medium with transferrinand antibiotics) was supplemented with EGF and insulin. In the resultingorganotypic culture on day 3, undifferentiated colonic epithelial cellsattached to the artificial stroma, but they were not polarized. On day7, in the presence of EGF and insulin, viable, non-polarized, epithelialcells survived in isolated small clusters with round-shapedundifferentiated morphology on the artificial stroma.

In other experiments, the growth factors, including insulin, EGF, SCF,ET-3, KGF, bFGF, or AMF were added individually to the base medium (MCDB201/L15 medium with transferrin and antibiotics). In each case,epithelial cell proliferation was not supported in the organotypicculture.

In still other experiments, base medium was supplemented with EGF,insulin, and LIF. By day 3, flat epithelial cells are migrating on theartificial stromal and form a monolayer covering the surface. On day 7,the colonic epithelial cells form highly cylindrical, polarizedepithelial clusters. Mesenchymal fibroblasts closely underline theepithelial layer, but goblet cells were absent.

In another experiment, the epithelial cells were cultured in base mediumsupplemented with HGF, AMF, insulin, and 1% FCS. The resultingorganotypic culture by day 4 had a flat, differentiated monolayer, whichcovers the entire artificial stroma.

In another experiment, the organotypic cultures were prepared by seedingepithelial cells on the artificial stroma in complete medium C or mediumL/S/E or medium L. Medium C contained transferrin, insulin, bFGF, EGF,ET-3, HGF, LIF, SCF, AMF, and 1% FCS as supplements. L/S/E mediumcontained transferrin, insulin, LIF, SCF, ET-3, EGF, and 1% FCS. Lmedium contained transferrin, insulin, LIF, EGF, and 1% FCS. Sections ofthe cultures were stained with H&E and Alcian blue after 12 hours (day0) and 4 days. Values were generated SD of 5 fields from 2 independentexperiments. The numbers of cells attached on day 0 between the threegroups were not significantly different. On day 4, using epithelial cellnumbers in complete medium C for comparison, the numbers of epithelialcells were significantly higher in L/S/E and L media (p<0.001). TheL/S/E-supplemented base medium (LIF, SCF, ET-3, EGF, insulin, and 1%FCS) induced formation by day 4 of highly disorganized structures ofstratified and pseudostratified epithelial cells. The epithelium washyperplastic with morphological atypia. In contrast to results obtainedwith complete growth medium, only the upper surface rather than theentire artificial stroma was covered with epithelium. The combination ofLIF, SCF, ET-3, EGF, insulin and 1% FCS stimulated formation of anepithelial layer with the highest cell numbers which were approximatelydouble compared to complete medium

This growth-inducing medium L/S/E stimulated the formation of gobletcells similar to those formed in complete growth medium. However, thenumber of goblet cells in L/S/E medium was significantly lower thanthose in complete medium (p<0.02) but not lower than those in C-medium(p<0.07).

EXAMPLE 3

Role of Extracellular Matrix (ECM)

Another embodiment of an organotypic culture prepared as describedsubstantially as in Example 2, has an additional component. Anextracellular matrix was applied to the organotypic cultures by coatingthe artificial stroma with matrix proteins Laminin-2 α2β1γ1 (LifeTechnologies, Rockville, Md.), Laminin-1 α2β1γ1 (Sigma), or Matrigel®gel matrix (Collaborative Research, Bedford, Mass.), which containsLaminin-1 and other matrix proteins, such as collagen IV and nitrogen(Burgeson, R E., et al. 1994 Matrix Biol., 14:209–211 and Page, KC. etal. 1990 J. Biol. Reprod., 43:659–664).

Fifty to 100 μl of base medium containing 20 ng/ml of laminins wereadded onto the surface of the artificial stroma for 60 minutes at 37° C.Matrigel® matrix was diluted 1:5 to 1:10 in base medium before use.After incubation, unbound laminins or Matrigel® matrix were removed bytwo washings with base medium. The organotypic cultures were incubatedat 37° C. in 5% CO₂ in growth medium, which was changed daily for 7days, then three times per week for an additional 21 days.

Growth and differentiation of colonic epithelium in growth medium wasregulated by ECM components at the interface between the stroma and theepithelial layer. These ECM experiments were done in base medium,supplemented with LIF, SCF, ET3, EGF, insulin, and 1% FCS. Coating ofthe artificial stroma (i.e., collagen and cellular) matrix with Matrigelmatrix and purified laminins increased adhesion and spreading. Theepithelial cells and clusters attached within 15 minutes and spreadwithin 30 to 60 minutes. When one portion of the artificial stroma wascoated with Matrigel matrix, which contains Laminin-1, and the otherwith purified Laminin-2, cells formed matrix-specific phenotypes.

Quantitative analysis of the growth fractions confirmed that thedifferent laminins control growth and differentiation. Laminin-1, eitheras purified matrix protein or when present in Matrigel matrix, induceddifferentiation, whereas Laminin-2 stimulated cell growth. Entericepithelial cells spread similarly on substrates of Laminins-2 and —Ibound to plastic of culture dishes. However, by day 5, the epithelialcells survived only with Laminin-2 as substrate and not with Laminin-1.The colorectal carcinoma cell line HT29 showed similarly better growthon Laminin-2 when compared to Laminin-1.

EXAMPLE 4

Organotypic Cultures of Normal Human Enteric Epithelium

The following experiments provide more recent data on the generation ofthese organotypic cultures of the invention.

A. Isolation Of Colonic Epithelial Cells.

Human enteric epithelial cells were isolated from fetal colon obtainedafter therapeutic or spontaneous abortions at 17–21 weeks' gestation.Specimens were received through Advanced Bioscience Resources (Alameda,Calif.) after approval by the Institutional Review Board. The colonlumen was opened and washed in Hank's buffered salt solution (HBSS;Gibco BRL, Rockville, Md.) supplemented with penicillin (200 U/ml),streptomycin (200 μg/ml) (Cellgro, Herndon, Va.) and gentamycin (100μg/ml) (Celigro). After removal of the serosa, the tissue was incubatedfor 10 minutes in HBSS containing 20 mg/ml Mucomyst®(N-acetyl-L-cysteine; Sigma, St. Louis, Mo.), pH 7.2, to removesurface-bound mucin. After three washings, tissue was dissociated at 37°C. in HBSS without Ca⁺⁺/Mg⁺⁺ (GIBCO BRL), supplemented with 1 mg/mlD-glucose (Sigma) and 1 mM ethylene diaminetetraacetic acid (EDTA;Sigma). Isolated colon mucosa was dissociated into single cells andsmall cell clusters. The epithelial layer was stripped, including thebottom of crypts.

After 10-minute incubation with occasional shaking, these single cellsand small cell clusters were removed and collected in a two-fold volumeof base medium supplemented with 5% fetal calf serum (FCS). Base mediumconsisted of 4 parts MCDB 201 medium and 1 part L15 medium (Sigma),supplemented with 2 ng/ml human recombinant EGF (Sigma), 5 μg/ml insulin(Sigma), 5 μg/ml transferrin (Sigma), 50 μg/ml streptomycin andgentamycin, respectively, and 2% FCS. The procedure was repeated twice.All samples were pelleted at 800×g for 10 minutes.

These single cells and cell clusters were washed and resuspended incomplete growth medium base medium supplemented with 10 ng/ml humanrecombinant basic fibroblast growth factor (bFGF) (Nesbit, M. et al,1999 Oncogene, 18, 6469–6476), 264 ng/ml endothelin-3 (ET-3; PeninsulaLabs, San Carlos, Calif.), 30 ng/ml hepatocyte growth factor (HGF; R&DSystems, Minneapolis, Minn.), 0.2 ng/ml LIF (R&D Systems), 30 ng/ml stemcell factor (SCF; Sigma), and 35 ng/ml autocrine motility factor (AMF;Sigma).

B. Isolation of Fibroblasts.

Human colonic fibroblasts were derived from colon explants from 17- to21-week fetuses. Fibroblasts of three specimens were cultured inDulbecco's modified minimum essential medium (DMEM; GIBCO BRL)supplemented with 10% FCS (Cansera, Rexdale, Ontario, Canada) andantibiotics. Cultures were used up to passage 10.

Human smooth muscle cells HIAS119, kindly provided by Dr. E. Levine, TheWistar Institute, were isolated from large blood vessels and maintainedin medium M199, supplemented with 10% FCS, 2 mM L-glutamine, and 50μg/ml bovine hypothalamus extract (Oda, D. et al, 1998 In Vitro Cell.Dev. Biol. Anim., 34, 46–52). Fibroblasts at 8×10⁵/ml were embedded incollagen type I (Organogenesis, Canton, Mass.) to a final concentrationof 0.9 to 1.1 mg/ml in DMEM supplemented with 50 μM vitamin C (Sigma),1.66 mM L-glutamine (GIBCO-BRL), and 1% FCS. The suspension wasneutralized, pH 7.2, using 7.5% sodium bicarbonate. Chambered slides,Lab-Tek 8 wells (Nunc International, Rochester, N.Y.), were filled with0.2 ml of the collagen and cell suspension. In initial experiments,fibroblasts were seeded together with smooth muscle cells (2×10⁴/ml).

C. Preparation of the Organotypic Culture

Colonic epithelial cells described above were seeded on top of collagengels containing embedded fibroblasts described above in 0.4 ml ofcomplete growth medium. After seeding onto the matrix of collagen type Iwith embedded fibroblasts, samples were incubated at 37° C. in 5% CO₂for 60–90 minutes in a low volume of medium (50–100 μl) to enhanceattachment. The epithelial cells and clusters attached within 60minutes. The wells were then filled with complete growth medium. Mediumwas changed daily for 10 days, then three times per week for anadditional 21 days.

D. Characterization of the Culture

Proliferation of organotypic cultures was determined by addingbromodeoxyuridine (BrdU) to the medium according to the manufacturer'sinstructions (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.).Thymidine incorporation was measured in cells, incubated with 1 μCi³H-thymidine/well for 18 hours before harvest and determination ofradioactivity.

The organotypic cultures were characterized as follows. Harvestedorganotypic cultures were fixed in 1.5% paraformaldehyde and embedded inparaffin. For electron microscopy (EM), cultures were fixed in 4%glutaraldehyde. For histochemistry and immunohistochemistry, 5-μm thicksections were cut from the paraffin beds and stained with hematoxylinand eosin (H&E) or Alcian Blue using standard procedures.

Goblet cells were detected using Alcian Blue and EM. Enteroendocrinecells were identified by chromogranin A staining and EM.Immunohistochemical staining was done with monoclonal antibodiesspecific for: BrdU (Amersham), Ki67 (DAKO, Carpinteria, Calif.),α-smooth muscle actin (SMA) (Sigma), cytokeratin 19 (CK 19; antibodyBA17 [33] kindly provided by Dr. J. Kovarik, Brno, Czech Rep.),chromogranin A (Novocastra, New Castle Upon Tyne, UK) and carbonicanhydrase II (The Binding Site Limited, Birmingham, UK).Immunohistochemistry was performed by standard techniques as described(Dai, C. Y. et al, 2000 Gastroenterology, 119, 929–942). Alkalinephosphatase activity of paraffin-embedded samples was detected usingcolorimetric substrate (AP Substrate Kit, SK 15100, Vector, Burlingame,Calif.).

Cell numbers were determined per high-power field (0.11×0.18 mm) using40× magnification as the mean (±SD) of 364 measurements. Constriction ofcollagen matrices was measured as the length of the longitudinal axis ofcollagen harvested on days 0.5, 4 and 10 (mean±SD of 32 measurements of4 samples. Positive cells per area were counted in 12 consecutive fieldsper cross-section (mean±SD of 16 areas of 2 random cross-sections from 4samples. Relative number of positive cells per area was expressed asmean±SD of positive/total cells from 16 areas of 2 random cross-sectionsfor each sample. Statistical significance was tested using Student'st-test.

In these cultures colonic epithelial cells attached within 60 minutes.The cells began spreading from clusters at 6 hours after seeding(FIG. 1) and migrated to cover the entire matrix. Constriction ofcollagen by the fibroblasts started after 24 hours and was maximal byday 4 when the matrix had shrunk to approximately 40% of its originalvolume (FIG. 1).

Because initial experiments with a mixture of fibroblasts and smoothmuscle cells (10:1) revealed no alteration of constriction or propertiesof the epithelial cells, all subsequent analyses were performed withfibroblasts only, which remained viable throughout the experiments. Asthe collagen matrix constricted, epithelial cell morphology changed fromflat during the initial migratory phase to polarized and columnar by day4. Mucin-producing goblet cells, based on their morphology and Alcianblue-positive staining, distributed throughout the epithelial celllayer. Fibroblasts migrated closely underneath the epithelial layer. The4 day reconstruct is partially covered by the epithelial layer. After 10days, epithelial cells covered all sides of the collagen matrix. Theepithelial layer was continuous and well-polarized. At this time, 2–10%of epithelial cells stained positive for the proliferation marker Ki67and up to 70% had incorporated BrdU when continuously added to themedium starting at the time of seeding. Total cell numbers began todecrease by day 20 when cells flattened. Mucin-producing goblet cells,identified by their typical round morphology and staining with AlcianBlue, were distributed throughout the epithelial cell layer. Thepercentage of goblet cells in the layers remained stable until day 10and individual cells had incorporated BrdU, but their total numberdecreased together with a decrease in all epithelial cells.

Fibroblasts remained dispersed throughout the collagen. Fibroblastsmigrating toward the epithelial cell layer expressed α-SMA as amyofibroblast marker. α-SMA-positive intestinal subepithelialmyofibroblasts were also seen in normal fetal human colon (control).Cells grown in monolayer or on a collagen matrix without fibroblasts didnot proliferate on day 4 as determined by ³H-thymidine incorporationassay and Ki67 immunohistochemistry, and they flattened and died by day8 at the latest.

EM analysis of the colonic epithelium in organotypic culture (10 day)revealed absorptive enterocytes with brush border, apical junctionalcomplexes with tight junction, desmosomes, interdigitating folds,immature basement membrane depositions and underlying mesenchymal cells,with rough endoplasmic reticulum and formation of regular brush border.Well-developed microvilli were observed on the apical surface of theepithelial cells. The upper lateral margins of all epithelial cells wereinterconnected by tight junctions. Intercellular adhesion complexes wereformed by desmosomes and interdigitating folds. Brush borders and tightjunctions were identified in mucin-producing goblet cells withmucin-containing granules. Fibroblasts closely underlay the epithelium,with immature basement membrane. Specific vesicles and lysosomes werelocated close to the base identified enteroendocrine cells, as confirmedby chromogranin A immunohistochemistry (see FIG. 6C). Between theepithelial and mesenchymal layers, an immature basement membranedeveloped. Fibroblasts, identified by their extensive rough endoplasmicreticulum, aligned below the basal side of the epithelial cells so thateach epithelial cell was underlined by a fibroblast. Such thin layers offibroblasts were absent in collagen surface areas free of epithelialcells. Neurosecretory cell containing specific vesicles and largersecondary lysosomes with underlying fibroblasts were also observed

Growth factors modulated epithelial cell growth and differentiation.Epithelial cell growth and differentiation in the organotypic culturewas achieved with complete growth medium of MCDB 201/L15 supplementedwith EGF, insulin, transferring, bFGF, ET-3, SCF, HGF, LIF, AMF, and 2%FCS. No single growth supplement sustained cell survival.

Base medium supplemented with EGF, insulin and transferring allowedsurvival of cells for up to 7 days. Additional growth factors in thebase medium altered morphology, differentiation and/or growth patternsof cells, inducing two distinct phenotypic patterns of the epithelialcells. Base medium containing EGF, insulin, transferring and 2% FCSsupplemented with HGF and AMF induced a flat, cuboidal cellularphenotype and the cells expressed the differentiation marker carbonicanhydrase II (identified by staining), which marker is found in theupper crypts and villi in normal colon. Base medium supplemented withLIF, ET-3, and SCF induces disorganized, multi-layered epithelium ofstratified and pseudo-stratified cells. The presence of LIF, ET-3, andSCF in the medium induced thickening of the colonic cell layer andinhibited expression of carbonic anhydrase II. Epithelial cells in bothgrowth media expressed cytokeratin 19, which is found throughout thenormal human colon, whereas alkaline phosphatase expressed in the uppercrypts of normal colon was not detected.

When only LIF was added to base medium (day 3), the cells flattened andmigrated to form a monolayer. On day 7, the proliferating epithelialcells show polarization with fibroblasts underlying the epitheliallayer. LIF-stimulated cultures formed disorganized structures ofstratified and semi-stratified cells that did not express thedifferentiation marker carbonic anhydrase II.

Control cultures maintained in the absence of LIF remained round, andpoorly attached by day 3, and cell numbers remained low, indicatinglittle proliferation by day 7. In the control cultures with nofibroblasts in the collagen matrix, LIF could not sustain epithelialcell survival, resulting in cell death. Epithelial cells did not survivein the absence of both LIF and fibroblasts.

Base medium with LIF and 2% FCS allowed migration of the epithelialcells around the entire matrix by day 7, whereas cells in the absence ofLIF covered only a portion of the matrix by day 7. Growth of epithelialcells was significantly stimulated by LIF (FIGS. 2A–2C). Cells showedsignificant incorporation of BrdU (indicating proliferation of bothfibroblasts in collagen and surface epithelial cells) and ³H-thymidine(FIG. 2A), with increased cell numbers/area (FIG. 2B). The stimulationof DNA replication and cell proliferation was solely attributable toLIF-mediated stimulation of epithelial cells and not of fibroblasts(FIG. 2C), LIF did not change collagen constriction by fibroblasts.

Double staining of organotypic cultures with BrdU and Alcian blueidentified differentiated cells in organotypic colon cultures, includingproliferating goblet cells. Goblet cells represented approximately 4% ofall enterocytes, but differences in relative numbers of LIF-culturedversus control base medium cultured cells were not significant (3.8±3.5%versus 4.2±3.2%, p=0.81) (FIG. 3A). There was a trend toward increasednumbers of goblet cells per area in LIF-stimulated samples (2.25±1.66versus 1.25±0.99 in samples without LIF, p=0.13). Cells cultured for 10days in the presence of BrdU and stained for Alcian blue showed thatapproximately 33% of the goblet cells had proliferated during this timeperiod. By contrast, relative numbers of enteroendocrine cells weresignificantly decreased in the presence of LIF (FIG. 3B). Cultureslabeled for 10 days with BrdU, stained and then counterstained withchromogranin A for detection of enteroendocrine cells revealed noproliferating enteroendocrine cells.

All references cited above as well as U.S. provisional patentapplication No. 60/314,111 are incorporated herein by reference.

1. An organotypic culture comprising an artificial stroma comprising amixture of collagen and human fibroblasts isolated from a human colon orintestine, said stroma overlayed with epithelial cells isolated fromfetal human colon, wherein said culture contains a growth factor thatbinds the insulin growth factor-1 (IGF-1) receptor selected from insulinand IGF-1, a growth factor that binds the epidermal growth factor (EGF)receptor selected from EGF and tumor growth factor-alpha (TGF-alpha),and the growth factor leukemia inhibitory factor (LIF), which binds theLIF receptor.
 2. The culture according to claim 1, wherein said collagenis selected from the group consisting of human collagen type I andbovine collagen type I.
 3. The culture according to claim 1, whereinsaid fibroblasts are selected from the group consisting of adult humancolon fibroblasts, fetal human colon fibroblasts, adult human smallintestinal fibroblasts, fetal human small intestinal fibroblasts, andhuman fibroblast stem cells.
 4. The culture according to claim 3,wherein said stem cells are isolated from human bone marrow.
 5. Theculture according to claim 1, further comprising human smooth musclecells embedded into said collagen.
 6. The culture according to claim 1,further comprising a layer of endothelial cells underlying said collagenand fibroblast mixture.
 7. The culture according to claim 1, comprisingat least one matrix protein selected from the group consisting ofLaminin-1 and Laminin-2.
 8. An organotypic culture comprising anartificial stroma comprising a mixture of collagen and human fibroblastsisolated from a human colon or intestine, said stroma overlayed withepithelial cells isolated from fetal human colon, wherein said culturecontains a growth factor that binds the IGF-1 receptor selected frominsulin and IGF-1, a growth factor that binds the EGF receptor selectedfrom EGF and tumor growth factor-alpha (TGF-alpha), the growth factorLIF, which binds the LIF receptor, the growth factor endothelin-3(ET-3), which binds the ET-3 receptor, a growth factor that binds thehepatocyte growth factor (HGF) receptor selected from HGF and tumorgrowth factor-beta (TGF-beta), the growth factor stem cell factor (SCF),which binds the SCF receptor, and the growth factor autocrine motilityfactor (AMF), which binds the AMF receptor.
 9. The culture according toclaim 8, further comprising an additional component selected from thegroup consisting of transferrin, fetal calf serum, and combinationsthereof.
 10. A method of preparing an organotypic culture of claim 1comprising: (a) assembling an artificial stroma by mixing collagen andfibroblasts; (b) seeding said artificial stroma with epithelial cellsisolated from fetal human colon in the presence of a growth factor thatbinds the IGF-1 receptor selected from insulin and IGF-1, a growthfactor that binds the EGF receptor selected from EGF and tumor growthfactor-alpha (TGF-alpha), and the growth factor LIF, which binds the LIFreceptor.
 11. The method according to claim 10, wherein said collagen isselected from the group consisting of human collagen type I and bovinecollagen type I.
 12. The method according to claim 10, wherein saidfibroblasts are selected from the group consisting of adult human colonfibroblasts, fetal human colon fibroblasts, adult human small intestinalfibroblasts, fetal human small intestinal fibroblasts, and humanfibroblast stem cells.
 13. The method according to claim 12, whereinsaid stein cells are isolated from human bone marrow.
 14. The methodaccording to claim 10, further comprising mixing human smooth musclecells into said collagen mixture.
 15. The method according to claim 10,further comprising layering said artificial stroma over a layer ofendothelial cells.
 16. The method according to claim 10, comprisingintroducing at least one matrix protein selected from the groupconsisting of Laminin-1 and Laminin-2 between said artificial stroma andsaid epithelial cells or overlaying said epithelial cells.
 17. Themethod according to claim 10, wherein said culture further comprises oneor more of a factor selected from the group consisting of endothelin-3,hepatocyte growth factor, stem cell factor and antocrine motilityfaetor.
 18. The method according to claim 10, wherein said culturefurther comprises an additional component selected from the groupconsisting of transferrin, fetal calf serum and combinations thereof.19. The method according to claim 10, further comprising seeding amalignant cell on the artificial stroma to create a model of tumorformation or tumor-stroma interaction.